Project Summary A forebrain structure, lamina terminalis (LT), plays a key role in both sensing internal water balance and regulating thirst through its downstream neural circuits. Recent studies have identified genetically-defined neural populations and circuit organization that control the initiation of drinking. The activity of these thirst neurons are rapidly suppressed with the onset of water consumption prior to absorption of ingested water. These results suggest that the LT integrates the homeostatic need and real-time satiety signals to optimize drinking. However, little is known about the functional significance of such integration and the underlying neural circuits. These studies have been hindered by the anatomical complexity of the LT and the lack of genetic handle on related neural circuits. Recent technological advances in transcriptomic analysis and neural manipulation/mapping tools have opened up an exciting window to study neural circuit at cell-type-specific precision. The present study combines such advanced approaches to delineate the cellular organization of the LT and the neural circuitry underlying rapid thirst satiety. These studies build on our results that thirst neurons in the subfornical organ (SFO) receive multiple satiety signals through anatomically and temporally separable neural substrates. In Aim 1, we will employ high-throughput single-cell RNA-seq analysis to elucidate a transcriptomic atlas of individual nuclei of the LT. This study will provide a framework to link individual physiological functions of the LT with molecularly-defined cell types. Based on our results, we will examine whether thirst neurons comprise functionally distinct multiple subpopulations in the LT. In Aim 2, we will characterize two temporally distinct satiety signals in the SFO induced by drinking action and osmolality change by water intake. We will determine the signaling pathways that carry individual thirst satiety signals using intragustric fluid infusion and in vivo optical recording from the SFO in awake-behaving animals. In Aim 3, we will define the neural substrates and circuits that mediate osmolality-induced satiety by retrograde viral tracing and electrophysiological tools. Once we identify candidate brain areas, we will apply an innovative ?monosynaptic? scRNA-seq analysis to identify specific genes enriched in the neurons that transmit the osmolality signal to the SFO. The outcome of this project will advance our understanding of neural basis of thirst and satiety regulation.